Microtubules are essential for many vital cellular processes, including formation of the mitotic spindle apparatus. The goal of our research is to identify and characterize molecular components of spindle poles, the organizing centers responsible for nucleating assembly of both mitotic and non-mitotic microtubules. Spindle pole proteins and other novel components that may interact with microtubules will be characterized using a combination of genetic, immunochemical, and biochemical approaches. The proposed experiments will utilize both Saccharomyces cercvisiae, which is readily amenable to genetic analysis and mammalian cells, in which certain cell biological experiments are more feasible. We will continue our analysis of SPAI, a yeast gene isolated with anti- spindle pole. To further understand its role in mitotic processes, a spal deletion mutant and a SPAI overproduction mutant will be constructed, and the effects of these mutations on cell viability, chromosome segregation, and other cellular processes will be studied. The effect of the deletion and overproduction mutations on spindle and spindle pole structure will be examined by indirect immunofluorescence and electron microscopy. Genetic and biochemical approaches will be initiated to identify additional genes important for spindle pole function in yeast. Genetic screens will be performed to search for mutants that have similar mitotic defects to spal cells. Extragenic suppressors of spa1 mutants will be sought, and those mutants with microtubule-associated defects will be studied further. Subcellular fractions containing the spindle pole body will be prepared in an attempt to identify and characterize other spindle pole components. We also plan to study the yeast SPA2 gene, which was isolated with human anti-spindle pole autoantibodies. The SPA2 protein has an interesting cytological location; it localizes to sites of cell growth which are also sites where cytoplasmic microtubules end. Cells with a disruption mutation in the SPA2 gene have defects in direction and control cell growth. The amino acid sequences that localize the SPA2 protein within the cell will be investigated. Genetic techniques will be used to identify proteins that interact with the SPA2 gene product. The study of spindle components will be extended to higher eukaryotic cells. We have already identified on potential cone encoding a human spindle pole protein; this clone will be characterized. The cell cycle expression of human spindle pole proteins will be analyzed. and role of these proteins in nucleating microtubule assembly will be examined.' The long term goal of these studies is to understand at a molecular level the components and events necessary for successful mitosis and cell growth.